NSAIDs induce both necrosis and apoptosis in guinea pig gastric mucosal cells in primary culture

A major clinical problem encountered with the use of nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin is gastropathy. In this study, we examined, using guinea pig gastric mucosal cells in primary culture, how NSAIDs damage gastric mucosal cells. The short-term treatment of cells with high concentrations of indomethacin decreased cell viability in the absence of apoptotic DNA fragmentation, chromatin condensation, or caspase activation. Cells lost membrane integrity with this short-term indomethacin treatment, suggesting that indomethacin induced necrosis under these conditions. In contrast, the long-term treatment of cells with low concentrations of indomethacin decreased cell viability and was accompanied by apoptotic DNA fragmentation, chromatin condensation, and caspase activation. Pretreatment of cells with inhibitors of caspases or protein synthesis suppressed cell death caused by long-term indomethacin treatment, suggesting that apoptosis was induced when the inhibitors were not present. These results imply that NSAIDs cause gastric mucosal damage through both necrosis and apoptosis of gastric mucosal cells.     

Helicobacter pylori lipopolysaccharide induces apoptosis of cultured guinea pig gastric mucosal cells

Helicobacter pylori lipopolysaccharide (LPS) is generally accepted as a low-toxicity virulence. Primary cultures of guinea pig gastric mucosal cells expressed the Toll-like receptor 4 and were sensitive to H. pylori LPS as well as Escherichia coli LPS. H. pylori LPS stimulated phosphorylation of transforming growth factor-beta-activated kinase 1 (TAK1), TAK1-binding protein 1 (TAB1), and c-Jun NH(2)-terminal kinase (JNK) 2. H. pylori LPS at >2.1 endotoxin unit/ml (>1 ng/ml) activated caspase-8, stimulated cytochrome c release from mitochondria, and subsequently activated caspases-9 and -3, leading to apoptosis. Epidermal growth factor blocked all of these apoptotic processes and inhibited apoptosis, whereas it did not modify the phosphorylation of TAK1, TAB1, and JNK2. A comparatively specific inhibitor of caspase-8 or -9 blocked apoptosis, whereas cytochrome c release was prevented only with a caspase-8-like inhibitor. Our results suggest that caspase-8 and mitochondria may play crucial roles in H. pylori LPS-induced apoptosis and that this accelerated apoptosis may be involved in abnormal cell turnover of H. pylori-infected gastric mucosa.     

Regulation of growth and apoptosis of cultured guinea pig gastric mucosal cells by mitogenic oxidase 1

We previously reported that primary cultures of guinea pig gastric pit cells expressed all of the phagocyte NADPH oxidase components (gp91-, p22-, p67-, p47-, and p40-phox) and could spontaneously release superoxide anion (O(2)(-)). We demonstrate here that pit cells express a nonphagocyte-specific gp91-phox homolog (Mox1) but not gp91-phox. Inclusion of catalase significantly inhibited [(3)H]thymidine uptake during the initial 2 days of culture. Pit cells, matured on day 2, slowly underwent spontaneous apoptosis. Scavenging O(2)(-) and related oxidants by superoxide dismutase plus catalase or N-acetyl cysteine (NAC) and inhibiting Mox1 oxidase by diphenylene iodonium activated caspase 3-like proteases and markedly enhanced chromatin condensation and DNA fragmentation. This accelerated apoptosis was completely blocked by a caspase inhibitor, z-Val-Ala-Asp-CH(2)F. Mox1-derived reactive oxygen intermediates constitutively activated nuclear factor-kappaB, and inhibition of this activity by nuclear factor-kappaB decoy oligodeoxynucleotide accelerated their spontaneous apoptosis. These results suggest that O(2)(-) produced by the pit cell Mox1 oxidase may play a crucial role in the regulation of their spontaneous apoptosis as well as cell proliferation.     

Ecabet sodium inhibits Helicobacter pylori lipopolysaccharide-induced activation of NADPH oxidase 1 or apoptosis of guinea pig gastric mucosal cells

Cholestatic hepatitis is frequently found in Niemann-Pick C (NPC) disease. We studied the influence of diet and the low density lipoprotein receptor (LDLR, Ldlr in mice, known to be the source of most of the stored cholesterol) on liver disease in the mouse model of NPC. Npc1-/- mice of both sexes, with or without the Ldlr knockout, were fed a 18% fat, 1% cholesterol ("high-fat") diet and were evaluated by chemical and histological methods. Bile acid transporters [multidrug resistance protein (Mrps) 1-5; Ntcp, Bsep, and OatP1, 2, and 4] were quantitated by real-time RT-PCR. Many mice died prematurely (within 6 wk) with hepatomegaly. Histopathology showed an increase in macrophage and hepatocyte lipids independent of Ldlr genotype. Non-zone-dependent diffuse fibrosis was found in the surviving mice. Serum alanine aminotransferase was elevated in Npc1-/- mice on the regular diet and frequently became markedly elevated with the high-fat diet. Serum cholesterol was increased in the controls but not the Npc1-/- mice on the high-fat diet; it was massively increased in the Ldlr-/- mice. Esterified cholesterol was greatly increased by the high-fat diet, independent of Ldlr genotype. gamma-Glutamyltransferase was also elevated in Npc1-/- mice, more so on the high-fat diet. Mrps 1-5 were elevated in Npc1-/- liver and became more elevated with the high-fat diet; Ntcp, Bsep, and OatP2 were elevated in Npc1-/- liver and were suppressed by the high-fat diet. In conclusion, Npc1-/- mice on a high-fat diet provide an animal model of NPC cholestatic hepatitis and indicate a role for altered bile acid transport in its pathogenesis.     

豚鼠肾小管上皮原代细胞的培养

目的建立一种简便易行的豚鼠原代肾小管上皮细胞培养方法。方法运用筛网分离法和多种酶消化法获取高纯度的肾小管上皮细胞。利用免疫组化法和形态学观察法鉴定培养的肾小管上皮细胞性质及纯度。结果通过肾小管节段贴壁,胶原酶消化组织节段和细胞等方法,有效地促进肾小管原代细胞增殖;胰酶节段消化法的细胞贴壁效果稍差,细胞传代状态不理想;胰酶消化法则细胞贴壁较少,细胞生长状态较差。结论培养豚鼠原代。肾小管上皮细胞是可行的。     

Phenylarsine oxide inhibits heat shock protein 70 induction in cultured guinea pig gastric mucosal cells

Phenylarsine oxide (PAO)forms a stable ring complex with vicinal dithiols that can be reversedwith 2,3-dimercaptopropanol (DMP) but not by dithiothreitol (DTT) or2-mercaptoethanol (2-ME). PAO at 2 µM or higher inhibited heat shockprotein 70 (HSP70) induction within minutes in cultured guinea piggastric mucosal cells exposed to heat (43°C) for 30 min. PAO did notaffect the nuclear translocation and phosphorylation of heat shockfactor 1 (HSF1) induced by heat stress, but it completely blocked the binding activity of HSF1 to the heat shock element (HSE), leading tothe block of expression of HSP70 mRNA and accumulation of HSP70 in thecells. These inhibitions were completely reversed with 2 µM DMP butnot with 0.1 mM DTT or 1 mM 2-ME, suggesting specific interactionsbetween PAO and vicinal dithiol-containing molecules. Thioredoxin (Trx)reversed the inhibition of the binding activity of HSF1 in whole cellextracts prepared from PAO-treated, heat-stressed cells. Our resultssuggest that PAO may react with vicinal-containing molecules includingTrx and specifically block the interaction between HSF1 and HSE.

     

Potassium transport in dispersed mucosal cells from guinea pig stomach

Dispersed mucosal cells (approx. 70% parietal cells) prepared from guinea pig stomach maintained their cellular concentration of potassium (65--80 nmol potassium/10(6) cells) for at least 5 h in vitro. Uptake of 42K by dispersed gastric mucosal cells depended on temperature, H+ concentration and oxidative metabolism. Carbachol and, in some instances, gastrin caused a 40--50% increase in cellular uptake of 42K as a consequence of the ability of these agents to increase 42K influx. Ouabain reduced uptake of 42K by 70% but did not alter the effect of carbachol. Cellular uptake of 42K was not altered by histamine, prostaglandin, E1, glucagon, secretin, vasoactive intestinal peptide or C-terminal octapeptide of cholecystokinin. Uptake of 42K was also increased by dibutyryl cyclic AMP or dibutyryl cyclic GMP but not by cyclic AMP, cyclic GMP or their 8-bromo derivatives. Theophylline caused a small (10--15%) increase in 42K uptake and potentiated the increase caused by submaximal concentrations of carbachol. The increase in 42K uptake caused by either dibutyryl cyclic nucleotide and carbachol was additive.     

A primary culture of guinea pig gallbladder epithelial cells that is responsive to secretagogues

We have developed a cell culture of guinea pig gallbladder epithelial cells with which to study ion transport. When grown on permeable supports, the cultured epithelia developed a transepithelial resistance (R(t)) of approximately 500 Omega. cm(2). The epithelial cell origin of the cell culture was further confirmed by immunocytochemical localization of cytokeratin. Ionomycin and forskolin increased transepithelial voltage and short-circuit current (I(sc)) and decreased R(t). The response to ionomycin was transient, whereas that to forskolin was sustained. Both were attenuated by replacement of Cl(-) and/or HCO(3)(-). Mucosal addition of the anion transport inhibitors DIDS or diphenylamine-2-carboxylic acid (DPC) blocked the response to ionomycin. The response to forskolin was blocked by DPC but not by DIDS. Ionomycin, but not forskolin, increased intracellular Ca(2+) concentration in fura 2-loaded cells. PGE(2), histamine, vasoactive intestinal polypeptide, and secretin elicited a sustained increase in I(sc). Responses to ATP and CCK were transient. Thus cultured guinea pig gallbladder epithelia display the range of responses observed in the native tissue and are an appropriate model for studies of ion transport in gallbladder and intestinal epithelia.     

Helicobacter pylori lipopolysaccharide enhances the expression of NADPH oxidase components in cultured guinea pig gastric mucosal cells.

Recently, we showed that cultured guinea pig gastric pit cells possess a phagocyte NADPH oxidase-like activity, which was up-regulated by Helicobacter pylori lipopolysaccharide. We demonstrate here that these cells express all of the phagocyte NADPH oxidase components (gp91-, p22-, p67-, p47-, and p40-phoxes). Treatment with lipopolysaccharide increased the expression of gp91-, p22-, and p67-phoxes, but not that of p47- and p40-phoxes. Intriguingly, the p67-phox expression consistently correlated with up-regulation of superoxide anion-producing ability. Thus, the gastric pit cell NADPH oxidase may play an important role in regulation of the inflammatory response associated with H. pylori infection.     

Aminopyrine uptake by guinea pig gastric mucosal cells. Mediation by cyclic AMP and interactions among secretagogues

The role of cyclic nucleotides in regulating acid secretion by dispersed mucosal cells from guinea-pig stomach was examined by measuring first the ability of histamine and carbachol to stimulate [dimethylamine-14C]aminopyrine uptake and cyclic nucleotide metabolism and secondly, the effect of exogenous cyclic nucleotides on basal and stimulated [14C]aminopyrine uptake. The [14C]aminopyrine was found in an acidic, osmotically sensitive compartment, probably associated with the initial steps in acid secretion by these cells. Although histamine increased [14C]aminopyrine uptake and cyclic AMP synthesis as expected, histamine was approx. 10-fold more potent in inducing [14C]aminopyrine uptake. This dissociation of [14C]aminopyrine uptake and cyclic AMP metabolism process was further manifested by the observation that prostaglandin E1 failed to increase [14C]aminopyrine uptake, although it did cause a rise in cellular cyclic AMP. Furthermore, prostaglandin E1 did not alter the [14C]-aminopyrine uptake caused by histamine. Carbachol was found to increase the [14C]aminopyrine uptake and also to potentiate the ability of histamine to increase [14C]aminopyrine uptake. Carbachol, however, affected neither the histamine-induced increase in cyclic AMP nor the binding of [3H]histamine to the cells. Cimetidine, a histamine H2 receptor antagonist, blocked the [14C]aminopyrine uptake induced either by histamine alone or by the potentiating combination of histamine plus carbachol. These results suggest that cyclic AMP is mediating the action of histamine on [14C]aminopyrine uptake but changes in cyclic AMP per se are not necessarily the cause for the potentiated increase in [14C]aminopyrine uptake. Furthermore, the potentiated response observed with histamine plus carbachol on [14C]aminopyrine uptake occurs at a biochemical step distal to and not obviously related to cyclic AMP generation.     

Cellular cyclic AMP in dispersed mucosal cells from guinea pig stomach.

In dispersed mucosal cells from guinea pig stomach cyclic AMP was increased 4-fold by theophylline, 5-fold by prostaglandin E2, and 10- to 15-fold by histamine. Theophylline augmented the increase in cellular cyclic AMP caused by histamine or prostaglandin E1 and the actions of histamine and prostaglandin E1 were additive. Cellular cyclic AMP was not altered by carbachol, gastrin, secretin, vasoactive intestinal peptide, glucagon, insulin or the octapeptide of cholecystokinin. Metiamide or diphenhydramine but not atropine inhibited the increase in cellular cyclic AMP caused by histamine, but did not alter the concentration of cyclic AMP in control cells or in cells incubated with theophylline or prostaglandin E1.     

Estrogen responsiveness of fetal guinea pig uterine cells in culture

Cells from the uterus of the guinea pig fetus have been grown as a monolayer culture in serum-containing medium. Cells from the first subculture showed high concentrations of progesterone receptor (PR; 9.3-13.8 pmol/mg DNA) even after 9 days in medium containing charcoal-treated serum and estradiol did not induce any further increase. The antiestrogens, tamoxifen and monohydroxytamoxifen, both had an inhibitory effect which could be overcome by estradiol. The progestins, progesterone and R5020, as well as the antiprogestin, RU38486, also decreased the PR concentration. Estrogen receptor (ER) levels did not vary with the compounds tested but were found to be low compared to concentrations found in the fetal guinea pig uterus at 55-65 days of gestation. None of the compounds tested had any effect on the growth of the fetal uterine cells so that the modulation of PR concentrations was dissociated from the regulation of cell growth. It is concluded that estrogens are necessary but not sufficient factors in the control of PR levels in fetal uterine cells. The establishment of a culture system for separate types of fetal uterine cells will permit us to study in vitro the factors involved in the growth effects of estrogens and the control of PR synthesis.     

Isolation and monolayer culture of guinea pig pancreatic duct epithelial cells

Summary Monolayers of cultured epithelial cells have been prepared from fragments of guinea pig pancreatic excretory ducts isolated by a simple procedure employing collagenase digestion and manual selection, through which virtually all of the ductal system can be recovered. The isolated fragments were cultured in enriched Waymouth's medium on extracellular matrices of various composition and thickness, including: thin (<5 μm) and thick (0.5 mm) layers of rat tail collagen; thin layers of human placental collagen; thin layers of Matrigel (a reconstituted basement membrane material); uncoated tissue culture plastic; and the cellulose ester membranes of Millipore Millicells. Cells spread rapidly from duct fragments cultured on uncoated plastic or on plastic coated with thin layers of rat tail collagen or human placental collagen and formed epithelial monolayers. However, these cells were squamous and lacked the abundant basolateral membrane amplification and apical microvilli characteristic of freshly isolated duct epithelial cells. Cells did not spread from duct fragments cultured on Matrigel. In contrast, when fragments of pancreatic ducts were explanted onto either a thick layer of rat tail collagen or onto Millicell membranes, cells readily spread and formed confluent monolayers of cuboidal epithelial cells characterized by abundant mitochondria, apical microvilli, and basolateral plasma membrane elaboration. These results demonstrate that different forms of extracellular matrix modulate the growth and differentiation of pancreatic duct epithelial cells, and that culture on a permeable substrate markedly enhances the maintenance of differentiated characteristics in this cell type. The monolayers formed on Millicell membranes should provide a useful model system for physiologic analysis of the regulation of electrolyte secretion by this epithelium. This research was supported by grants DK32994 and DK35912 from the National Institutes of Health, Bethesda, MD.     

Regulation of c-fos expression in primary culture of guinea pig glandular epithelial cells stimulated by growth factors and estradiol

The c-fos expression was investigated in primary culture of guinea pig glandular epithelial cells. These cells were made quiescent by serum deprivation and stimulated with fetal calf serum (FCS, 15%), 17 beta-estradiol (E2 10(-8) mol/l) alone or in combination with epidermal growth factor (EGF, 100 ng/ml) and insulin (I, 10 micrograms/ml). Low levels of c-fos mRNA were detectable in quiescent cells and were not increased in cells stimulated with either E2, EGF, I, or EGF plus I. On the contrary, the c-fos mRNA were early and transiently increased by FCS or E2 plus EGF plus I (4.5 and 9.5 fold induction, respectively). This effect was independent of de novo protein synthesis since it was not abolished in the presence of cycloheximide. It appears that E2 acts in a multiple step process including the stimulation by EGF plus insulin.     

Pepsinogen synthesis in monolayer culture of human and rabbit gastric mucosal cells

We have established monolayer cultures of human and rabbit gastric mucosal cells and of isolated rabbit gastric chief cells. These cultures were capable of de novo pepsinogen synthesis and secretion, demonstrated by electrophoresis and subsequent autoradiography of cell lysates and growth medium after culture in the presence of 14C-labelled amino acids. Cultures could be maintained for 1 week without overgrowth by fibroblasts.     

Visualization of cytosolic free calcium distribution and mobilization in guinea pig gastric chief cells

Distribution and temporal change of free calcium concentration [( Ca2+]i) in single guinea pig gastric chief cells were visualized by a digital imaging microscope equipped with a microspectrofluorometer. The distribution was not homogeneous; a higher [Ca2+]i area was often localized in some restricted regions of the endoplasm and also at the peripheral cytoplasm just beneath the plasma membrane. When stimulated with cholecystokinin, [Ca2+]i increased transiently in the apical peripheral cytoplasm and in the endoplasmic regions. This Ca2+ mobilization which precedes the biphasic pepsinogen secretion was composed of a rapid Ca2+ release from the intracellular store(s) as well as a rapid and a more sustained Ca2+ entry from the extracellular space.     

Contribution of spermidine to stimulation by hepatocyte growth factor in repair after damage of rabbit gastric mucosal cells in primary culture

Wound-healing of the gastric mucosa is suggested to be stimulated by hepatocyte growth factor (HGF). Polyamines are shown to contribute to repair after damage in the gastric mucosa. The present study was designed to elucidate whether HGF can stimulate wound-healing of the gastric mucosa via polyamine production, using rabbit gastric mucosal cells in primary culture. A wound was made as a round cell-free area in the cell sheet of confluent cultured cells. When HGF was added to the culture medium, such denuded area was significantly reduced in size compared with the control, but the reduction was inhibited by addition of D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of a rate-limiting enzyme (ornithine decarboxylase) of polyamine biosynthesis, to the culture medium. However, the inhibitory effect by DFMO was reversed by pretreatment with spermidine, but not with putrescine. Intracellular levels of polyamines in the whole confluent cells including the cells around the denuded area were not changed by addition of HGF, but putrescine and spermidine levels were decreased by further addition of DFMO. We conclude that spermidine may be involved in stimulation by HGF in the repair after damage of gastric mucosal cells.